Although a large number of about 20-26 kDa guanine nucleotide-binding proteins with sequence homology to the protooncogene ras have been identified by cDNA cloning, only some of the corresponding proteins have been purified and characterized. Recently it was found that members of this ras superfamily, rhoA, B, and C and rac1 and 2 are substrates for Clostridium botulinum C3 ADP-ribosyltransferase (C3). The functions of rho and rac have not been defined, although injection of C3 may lead to effects similar to those observed with activated ras. Two C3 substrates were purified from bovine brain cytosol and identified by microsequencing. The partial amino acid sequences were identical to the deduced amino acid sequences of rhoA and rhoB, respectively and hence they are referred to as rhoA* and rhoB*. rhoB* is the first identified soluble form of rhoB; it exhibits properties similar to those reported for membrane-associated rhoB. rhoA* has characteristics clearly different from other rhoA-like proteins. rhoA* migrates as a 77-80 kDa protein on gel filtration; however on SDS-PAGE, the ADP-ribosylated protein has a mobility consistent with a protein of 21.5 kDa. C3-catalyzed ADP-ribosylation of rhoA* was dependent on guanine nucleotides even in 1 mM Mg2+. Half-maximal stimulation by GTP, GTP-(gamma)S, Gpp(NH)p, and GDP was observed at 16, 20, 220, and 380 nM, respectively. GD(beta)S, GMP and adenine nucleotides were ineffective. Additionally, in the presence of guanine nucleotide, the rate and extent of ADP-ribosylation were enhanced by dimryistylphophatidylcholine (DMPC) and cholate. These findings are consistent with the possibility the rhoA* is isolated as part of a complex in which it exhibits sensitivity to guanine nucleotides. The complex could be self-associated rhoA* or rhoA* tightly associated with a cytosolic protein in a manner that makes ADP-ribosylation by C3 sensitive to guanine nucleotides and phospholipids.